The "HAT-TRICK" technique: A modification of soft tissue grafting using volume stable collagen matrix and cross-linked hyaluronic acid. Part A: The pontic site.

Vertical soft tissue augmentation between implants can be clinically challenging and burdensome for patients when employing conventional techniques. Recently, with the introduction of xenogenic collagen matrices, the principle of single-site surgery has become more common. However, some issues persist regarding graft stability and tissue integration. In the present technical note, the authors introduce the "HAT-TRICK" technique to address these observed difficulties. As the name suggests, this technique is believed to provide improved stability, volumetric gain, and histological integration of the implanted matrix by shaping it appropriately resembling a hat over the crest with apical bevels, stabilized with fixation pins and infused with cross-linked hyaluronic acid (xHya). A two-month observation of a bi-maxillary case is presented with detailed description of the technique and digitalized comparison methods for an easier explanation of the introduced technique.

Cytotoxicity of V-Prep Versus Phosphoric Acid Etchant on Oral Gingival Fibroblasts.

The most used etchant in dental daily practice is the phosphoric acid (P.A.; 37%). However, acid etchants can induce necrosis on the oral mucosa and cause the ulceration of periodontal tissue when a rubber dam is not used. V-prep is a new practical alternative, and it has satisfactory results. It is used as a preparation before the application of a resin-modified glass ionomer composite (RMGIC) to bond the orthodontic brackets. The aim of this study was to investigate the effect of the V-prep on oral gingival fibroblasts cells by comparing the cell damage and cell viability after the use of V-prep and a conventional phosphoric acid etchant with different application times and concentrations. Therefore, Gingival fibroblasts passage 6 (GFP6) was grown and treated with an acid etchant and V-prep at three different concentrations (1:1, 1:2 and 1:10) for two different application durations (30 s and 1 min). The morphological changes, cell death and cell viability were assessed. Pyknosis, karyolysis, nucleus reversible and irreversible damages and membrane destruction were observed for both of the etchants at the higher concentrations and longer application durations. Mann-Whitney U-tests were used for the statistical analyses. The application of the V-prep for 30 s showed better values than the acid etchant did in the cell damage analysis and cell viability analysis (p = 0.03). V-prep at a 1:10 concentration applied for a 30 s duration can preserve the viability of gingival fibroblasts cells up to 100%. The toxicity of V-prep is equal or lower than the toxicity of the acid etchant that is commonly used in dentistry. Thus, the V-prep can be used with precautions intra-orally, and it should be applied on the enamel as a gel for 30 s only before it is rinsed and removed.

Inherited connective tissue diseases highlight macromolecular network interdependences in skin extracellular matrix: a histomorphometric study.

Mutation of just a single extracellular matrix protein, a receptor or enzyme involved in connective tissue metabolism is sufficient to cause systemic pathologies and failure of tissues that are subjected to strong mechanical stresses. Skin histological and computerized image analyses can provide a good qualitative and quantitative indication of these inherited connective tissue diseases. In this study, skin biopsies from young (10 to 25 years) and middle-aged patients (26 to 50 years) suffering from Ehlers-Danlos syndromes (EDS), Marfan syndrome (MS) or pseudoxanthoma elasticum (PXE) were studied after specific staining of both the collagen and elastic networks. Findings from the histomorphometric analyses conducted on skin sections of the patients with EDS, MS and PXE were compared to skin sections of healthy subjects from the same age groups. Our results show that both the collagen and the elastic networks were affected in all the studied pathological cases, but that the adverse changes to the elastic network in older patients were distinct from the physiological changes observed during aging process for healthy subjects. This degenerative process may be explained by an added phenomenon involving a general connective tissue proteolysis.

Oral manifestations associated with inherited hyperhomocysteinemia: A first case description.

Hyperhomocysteinemia is a rare disease caused by nutritional deficiencies or genetic impairment of cysteine metabolism. To date, no oral manifestations of hyperhomocysteinemia have been described in humans. Therefore, to our knowledge, the present case report is the first description of a hyperhomocysteinemic patient showing oral tissue alterations leading to both early tooth loss and failed implant osseointegration. The patient presented with a methylenetetrahydrofolate reductase gene mutation (677T polymorphism) leading to mild hyperhomocysteinemia. The radiologic analysis showed hyperdense lesions scattered in the maxillae. The histologic observations indicated alterations in both collagen and elastic networks in the gingiva and dermis. Interestingly, the presence of ectopic mineralized inclusions was noted in both periodontal ligament and gingiva. Strong osteoclastic activity was associated with abnormal calcification of trabecular spaces. Uneven oral tissue remodeling due to high tissue levels of homocysteine could explain the pathologic manifestations observed in this case.

Poor Biointegration of Porcine Acellular Dermal Matrix Associated with Unfavorable Gingival Healing: A Report of Three Cases.

AIM: The aim of the present work was to explain the poor biointegration of acellular dermal xenogeneic matrix, leading to an unfavorable gingival healing following a grafting procedure for the treatment of soft tissue deficiencies. BACKGROUND: Numerous works have demonstrated the successful use of acellular dermal matrix (ADM) in soft tissue augmentation procedures. However, spare human investigations reported adverse healing outcomes at microscopic level. CASE DESCRIPTION: Three patients showing various soft tissue deficiencies (recession, gingival thickening) requiring a gingival augmentation were grafted using an ADM porcine acellular dermal matrices (pADM) as a soft tissue substitute. For this purpose, appropriate soft tissue augmentation surgeries were performed and the grafted pADM was left for proper healing. Biopsies were harvested from two out of the three patients, respectively, at 11 and 27 weeks in order to conduct a histological evaluation of the pADM's doubtful biointegration. Moreover, the ultrastructural analysis of pADM was performed using scanning electron microscopy, and additional histological procedures were used to assess its ability to support human gingival fibroblast cultures. Signs of gingival inflammation persisted several months postoperatively. Histologically, numerous inflammatory cells characterized the grafted site. Indeed, the high number of foreign body giant cell granulomas and the very densified newly formed collagen fibers highlighted a fibrotic process within gingival connective tissue. The ultrastructural and histological analysis showed that pADM was characterized by very thick and dense collagen bundles demonstrating a nonphysiological collagen network organization. Cell culture experiments showed fibroblasts proliferating on the matrix surface, sparing its deeper part, even though the collagen matrix degradation seemed to occur following a gradient from the pADM surface inward. CONCLUSION: The unfavorable clinical results may be caused by the poor colonization of matrix cells and poor angiogenesis leading to the inadequate biointegration of pADM. Hence, the pADM structure in terms of porosity and degradability should be further investigated. CLINICAL SIGNIFICANCE: The present cases highlighted a poor integration of pADM following soft tissue grafting procedures, which was caused by the inadequate ultrastructure of the used pADM. Therefore, despite the utility of such tissue substitutes, their manufacturing improvement could be required to obtain a better biointegration.

Engineering of Bio-Adhesive Ligand Containing Recombinant RGD and PHSRN Fibronectin Cell-Binding Domains in Fusion with a Colored Multi Affinity Tag: Simple Approach for Fragment Study from Expression to Adsorption.

Engineering of biomimetic motives have emerged as promising approaches to improving cells' binding properties of biomaterials for tissue engineering and regenerative medicine. In this study, a bio-adhesive ligand including cell-binding domains of human fibronectin (FN) was engineered using recombinant protein technology, a major extracellular matrix (ECM) protein that interacts with a variety of integrins cell-surface's receptors and other ECM proteins through specific binding domains. 9th and 10th fibronectin type III repeat containing Arginine-Glycine-Aspartic acid (RGD) and Pro-His-Ser-Arg-Asn (PHSRN) synergic site (FNIII9-10) were expressed in fusion with a Colored Multi Affinity Tag (CMAT) to develop a simplified production and characterization process. A recombinant fragment was produced in the bacterial system using E. coli with high yield purified protein by double affinity chromatography. Bio-adhesive surfaces were developed by passive coating of produced fragment onto non adhesive surfaces model. The recombinant fusion protein (CMAT-FNIII9/10) demonstrated an accurate monitoring capability during expression purification and adsorption assay. Finally, biological activity of recombinant FNIII9/10 was validated by cellular adhesion assay. Binding to α5ß1 integrins were successfully validated using a produced fragment as a ligand. These results are robust supports to the rational development of bioactivation strategies for biomedical and biotechnological applications.

In vitro and in vivo proves of concept for the use of a chemically cross-linked poly(ester-urethane-urea) scaffold as an easy handling elastomeric biomaterial for bone regeneration.

Bone loss can occur as a result of various pathologies, traumas and injuries and poor bone healing leads to functionally debilitating condition, loss of self-sufficiency and deterioration in life quality. Given the increasing incidence of facial trauma and the emergence of new procedural techniques, advanced scaffolds are currently developed as substitutes for bone tissue engineering. In this study, we investigated the capability of a chemically cross-linked ε-caprolactone-based poly(ester-urethane-urea) (PCLU) scaffold to support bone regeneration. In vitro assays demonstrated that PCLU scaffolds could be colonized by cells through direct cell seeding and cell migration from outside to scaffold inside. Moreover, PCLU scaffolds could provide a suitable environment for stem cells proliferation in a 3D spatial arrangement, and allowed osteogenic differentiation under appropriate induction. In vivo results revealed the osteogenic properties of PCLU scaffolds through a drilled-hole femoral bone defect repair improvement in rats. Using histology and microtomography analysis, we showed that PCLU scaffolds fit well the bone cavity and were eventually entrapped between the newly formed trabeculae. Finally, no sign of inflammation or rejection was noticed. We envision that PCLU scaffolds can provide the clinicians with a substitute having appropriate characteristics for the treatment of bone defects.

The Use of Platelet-Rich Plasma to Promote Cell Recruitment into Low-Molecular-Weight Fucoidan-Functionalized Poly(Ester-Urea-Urethane) Scaffolds for Soft-Tissue Engineering.

Due to their elastomeric behavior, polyurethane-based scaffolds can find various applications in soft-tissue engineering. However, their relatively inert surface has to be modified in order to improve cell colonization and control cell fate. The present study focuses on porous biodegradable scaffolds based on poly(ester-urea-urethane), functionalized concomitantly to the scaffold elaboration with low-molecular-weight (LMW) fucoidan; and their bio-activation with platelet rich plasma (PRP) formulations with the aim to promote cell response. The LMW fucoidan-functionalization was obtained in a very homogeneous way, and was stable after the scaffold sterilization and incubation in phosphate-buffered saline. Biomolecules from PRP readily penetrated into the functionalized scaffold, leading to a biological frame on the pore walls. Preliminary in vitro assays were assessed to demonstrate the improvement of scaffold behavior towards cell response. The scaffold bio-activation drastically improved cell migration. Moreover, cells interacted with all pore sides into the bio-activated scaffold forming cell bridges across pores. Our work brought out an easy and versatile way of developing functionalized and bio-activated elastomeric poly(ester-urea-urethane) scaffolds with a better cell response.

Characterization of elastomeric scaffolds developed for tissue engineering applications by compression and nanoindentation tests, µ-Raman and µ-Brillouin spectroscopies.

In tissue engineering, porous biodegradable scaffolds are developed with morphological, chemical and mechanical properties to promote cell response. Therefore, the scaffold characterization at a (sub)micrometer and (bio)molecular level is paramount since cells are sensitive to the chemical signals, the rigidity, and the spatial structuring of their microenvironment. In addition to the analysis at room temperature by conventional quasi-static (0.1-45 Hz) mechanical tests, the ultrasonic (10 MHz) and µ-Brillouin inelastic light scattering (13 GHz) were used in this study to assess the dynamical viscoelastic parameters at different frequencies of elastomeric scaffolds. Time-temperature superposition principle was used to increase the high frequency interval (100 MHz-100 THz) of Brillouin experiments providing a mean to analyse the viscoelastic behavior with the fractional derivative viscoelastic model. Moreover, the µ-Raman analysis carried out simultaneously during the µ-Brillouin experiment, gave the local chemical composition.

Cytotoxic evaluation of a new ceramic-based root canal sealer on human fibroblasts.

OBJECTIVES: The purpose of this study was to evaluate the cytotoxicity of a new bioceramic-based root canal sealer (BioMM) by direct and indirect contact with human fibroblasts and to compare it with a zinc oxide-eugenol sealer, the Pulp Canal Sealer-extended working time (PCS-EWT). MATERIALS AND METHODS: Cell viability was assessed through direct and indirect contact between human fibroblasts and sealer. Direct contact was performed at 24 h, whereas the indirect contact was performed at 24 and 48 h at different concentrations: 100%, 50%, and 25%. After direct contact, 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used and the optical density was measured by a spectrophotometer. Giemsa stain was also performed for a qualitative evaluation of the cells. STATISTICAL ANALYSIS USED: Shapiro-Wilk test was used to verify the normality of distribution of the variable. Data were analyzed by analysis of variance and Tukey tests using SPSS for Windows software 18.0. The significance level used was P < 0.05. RESULTS: Direct contact showed a significantly higher cell viability with BioMM as compared to PCS-EWT (P = 0.002). Cell viability at 24 h was significantly higher with BioMM compared to PCS-EWT for the concentrations of 50% (P = 0.004) and 25% (P = 0.003), whereas no significant difference was noted at 100% (P = 0.141). Cell viability at 48 h was significantly higher with BioMM as compared to PCS-EWT at 25% (P = 0.007). No significant difference was observed at 100% (P = 0.484) and 50% (P = 0.185). CONCLUSION: BioMM may be considered minimally cytotoxic if accidentally extruded into the periapical tissues.

Preliminary In Vitro Assessment of Stem Cell Compatibility with Cross-Linked Poly(ε-caprolactone urethane) Scaffolds Designed through High Internal Phase Emulsions.

By using a high internal phase emulsion process, elastomeric poly(ε-caprolactone urethane) (PCLU) scaffolds were designed with pores size ranging from below 150 µm to 1800 µm and a porosity of 86% making them suitable for bone tissue engineering applications. Moreover, the pores appeared to be excellently interconnected, promoting cellularization and future bone ingrowth. This study evaluated the in vitro cytotoxicity of the PCLU scaffolds towards human mesenchymal stem cells (hMSCs) through the evaluation of cell viability and metabolic activity during extract test and indirect contact test at the beginning of the scaffold lifetime. Both tests demonstrated that PCLU scaffolds did not induce any cytotoxic response. Finally, direct interaction of hMSCs and PCLU scaffolds showed that PCLU scaffolds were suitable for supporting the hMSCs adhesion and that the cells were well spread over the pore walls. We conclude that PCLU scaffolds may be a good candidate for bone tissue regeneration applications using hMSCs.

Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues.

Specific staining of the extracellular matrix components is especially helpful in studying tissue remodeling, particularly in the case of connective tissue pathologies. As developed by Junqueira and colleagues in 1979, specific staining by Picrosirius red is one of the most important stains to study collagen networks in different tissues. Under polarized light, collagen bundles appear green, red or yellow, and are easily differentiated from the black background, thus allowing for quantitative morphometric analysis. As Junqueira and colleagues point out, many studies use color staining to differentiate collagen bundles and to specify collagen types, yet other studies report that polarized colors only reflect fiber thickness and packing. Using a simple histological example, our study illustrates the inability of Picrosirius red staining to differentiate collagen types, since the absorbed amount of polarized light by this dye strictly depends on the orientation of the collagen bundles.

PolyNaSS bioactivation of LARS artificial ligament promotes human ligament fibroblast colonisation in vitro.

BACKGROUND: Introduction of a new generation of artificial ligaments for ACL reconstruction, the Ligament Augmentation and Reconstruction System (LARS), gives promising clinical results [1]. The current literature supports the use of LARS from short to medium term. To go even further to improve the biocompatibility of this biomaterial, poly(sodium styrene sulfonate) (polyNaSS) was grafted onto its surface. Studies using sheep animal model showed improvement of knee functionalities with this grafted artificial ligament and a better adhesion of human cell lines. OBJECTIVES: To better understand this in vivo improvement of integration with the bioactivated artificial prosthesis, in vitro studies were leaded using human ligament fibroblasts. METHODS: Human ligament fibroblasts isolated from human ruptured ACL were amplified and seeded onto poly(NaSS) grafted and non-grafted PET scaffold (Lars ligament) under standard culture conditions. Cellularized fibers were observed under scanning electron microscopy and histological and immunohistological studies were performed. RESULTS: Cells are localized around the grafted PET fibers of the bioactive ligament and penetrate in the scaffold. On ungrafted fibers, cells stay around the scaffold. On grafted fibers, collagen I appears strongly organized whereas is thin and dispersed on non grafted fibers. Finally, grafting altered localization of decorin. CONCLUSIONS: PolyNaSS grafting enhances human ligament fibroblast organisation in vitro in contact with biomaterial and improves collagen and decorin deposits around fibers.

Unusual glycosaminoglycans from a deep sea hydrothermal bacterium improve fibrillar collagen structuring and fibroblast activities in engineered connective tissues.

Biopolymers produced by marine organisms can offer useful tools for regenerative medicine. Particularly, HE800 exopolysaccharide (HE800 EPS) secreted by a deep-sea hydrothermal bacterium displays an interesting glycosaminoglycan-like feature resembling hyaluronan. Previous studies demonstrated its effectiveness to enhance in vivo bone regeneration and to support osteoblastic cell metabolism in culture. Thus, in order to assess the usefulness of this high-molecular weight polymer in tissue engineering and tissue repair, in vitro reconstructed connective tissues containing HE800 EPS were performed. We showed that this polysaccharide promotes both collagen structuring and extracellular matrix settle by dermal fibroblasts. Furthermore, from the native HE800 EPS, a low-molecular weight sulfated derivative (HE800 DROS) displaying chemical analogy with heparan-sulfate, was designed. Thus, it was demonstrated that HE800 DROS mimics some properties of heparan-sulfate, such as promotion of fibroblast proliferation and inhibition of matrix metalloproteinase (MMP) secretion. Therefore, we suggest that the HE800EPS family can be considered as an innovative biotechnological source of glycosaminoglycan-like compounds useful to design biomaterials and drugs for tissue engineering and repair.

Clinical, histological, and histomorphometrical analysis of maxillary sinus augmentation using inorganic bovine in humans: preliminary results.

The aim of the present study was to evaluate bone formation after maxillary sinus augmentation using bovine bone substitute material Bio-Oss alone by means of clinical, histological, and histomorphometrical examination of human biopsies. Deproteinized bovine bone (DPBB, Bio-Oss) was used to fill cavities after elevation of the sinus mucosa following major sinus pneumatization. Twenty patients with edentulous posterior maxillae were treated with 20 sinus augmentation procedures using a 2-stage technique. Residual lateral maxillary bone height was less than 3 mm. Forty-nine Straumann endosseous implants were used to complete the implant-prosthetic rehabilitation. Forty cylinder-shaped bone biopsies were taken from the augmented maxillary region 8 months after grafting during the second-stage surgery before implant placement. All implants were loaded 3 months after insertion, and no failures were recorded. Histomorphometrical analysis showed an average percentage of newly formed bone of 17.6% (± 2.8%) and a proportion of residual bone substitute material of 29.9% (± 4.9%) of the total biopsy area. Intimate contact between newly formed bone and Bio-Oss was detected along 28.2% (± 6.8%) of the particle surfaces. The results also showed that in all cases, the DPBB granules had been interconnected by bridges of vital newly formed bone. Inorganic bovine bone appears to be biocompatible and osteoconductive, and it can be used with success as a bone substitute in maxillary sinus augmentation procedures.

Epigallocatechin gallate's protective effect against MMP7 in recessive dystrophic epidermolysis bullosa patients.

The analysis of phenotype-genotype correlations of patients suffering from recessive dystrophic epidermolysis bullosa (RDEB) evidenced intrafamilial and interfamilial phenotype variability occurring for the same mutation of COL7A1; this underscores the role of other genetics environmental factors in the expressivity of the disease. In this work, we checked whether matrilysin 1 (matrix metalloproteinase (MMP)7) could take part in the epidermal detachment in RDEB. Furthermore, we investigated epigallocatechin 3 gallate (EGCG) to determine whether it could inhibit matrilysin activities on collagen type VII and fibrillin 1 known to be associated with the dermo-epidermal junction. In this work, matrilysin 1 was detected in affected and unaffected skins of the three RDEB patients; furthermore, MMP7 was shown to degrade ex vivo on healthy normal skin collagen VII and fibrillin 1. Thus, we suspect that MMP7 could take an active part in the epidermal detachment occurring during RDEB. We evidenced that EGCG in in vitro as well as in ex vivo experiments was a good inhibitor of MMP7 and developed a good protection of collagen type VII and fibrillin 1 susceptible of being degraded by MMP7. We therefore propose that EGCG could be used beneficially in patients suffering from RDEB.